The present invention relates, in general, to vascular smooth muscle proliferation and, in particular, to a method of inhibiting arterial and venous smooth muscle proliferation resulting, for example, from arterial injury or vein grafting. The invention also relates to an expression construct encoding a Gxcex2xcex3 inhibitor suitable for use in such a method.
Several growth factors that induce cellular mitogenesis and proliferation act through membrane-embedded G protein-coupled receptors (GPCRs). GPCRs couple to, and stimulate, heterotrimeric G proteins which, upon activation, dissociate to Gxcex1 and Gxcex2xcex3 subunits. Both these molecules can transduce intracellular signals via activation of specific effect or proteins. The intracellular signaling events leading to cellular proliferation following GPCR-activation appear to be transduced largely through the activation of p21ras (Ras) and subsequent activation of the p42 and p44 mitogen-activated protein (MAP) kinases. Growth factors which act through GPCRs, such as lysophosphatidic acid (LPA) via the LPA receptor and norepinephrine via xcex12-adrenergic receptors, have been shown to activate Ras and MAP kinase primarily through Gxcex2xcex3 (Koch et al, Proc. Natl. Acad. Sci. USA 91:12706 (1994)).
The last 194 amino acids (Gly495-Leu689) of the bovine xcex2-adrenergic receptor kinase-1 (xcex2ARK-1) represent a specific and selective Gxcex2xcex3-inhibitor (see FIG. 1 for amino acid sequence of xcex2ARK-1-(495-689) and a nucleic acid sequence encoding same). xcex2ARK-1 is a Gxcex2xcex3-dependent, cytosolic enzyme which must translocate to the membrane where it can phosphorylate its receptor substrate by physically binding to the membrane-anchored Gxcex2xcex3 (Pitcher et al, Science 257:1264 (1992)). 
The peptide encoded by the plasmid designated xcex2ARK-1-(495-689) Minigene (which peptide is designated xcex2ARKCT) contains the specific Gxcex2xcex3-binding domain of xcex2ARK-1 (Koch et al, J. Biol. Chem. 268:8256 (1993)). When cells are transfected with the xcex2ARK-1-(495-689) Minigene (that is, the xcex2ARKCT Minigene), or peptides containing the Gxcex2xcex3-binding domain of xcex2ARK-1 are introduced into cells, several Gxcex2xcex3-dependent processes are markedly attenuated including xcex2ARK-1-mediated olfactory receptor desensitization (Boekhoff et al, J. Biol. Chem. 269:37 (1994)), phospholipase C-xcex2 activation (Koch et al, J. Biol. Chem. 269:6193 (1994)) and Gxcex2xcex3-dependent activation of Type II adenylyl cyclase (Koch et al, Biol. Chem. 269:37 (1994)). These studies demonstrate that the xcex2ARK-1-(495-689) peptide (that is, xcex2ARKCT) is Gxcex2xcex3-specific, that is, that it does not alter Gxcex1-mediated responses (Koch et al, Proc. Natl. Acad. Sci. USA 91:12706 (1994); Koch et al, Biol. Chem. 269:37 (1994)). A further study utilizing the xcex2ARKCT Minigene has demonstrated that the growth factor IGF-1, by binding to its specific receptor, activates the Ras-MAP kinase pathway via Gxcex2xcex3. These results indicate that certain receptor-tyrosine kinase-mediated cascades include a Gxcex2xcex3 component, as do those for LPA and other agonists that activate classical GPCRs (Luttrell et al, J. Biol. Chem. 270:16495 (1995)).
The present invention is based, at least in oart, on the observation that the xcex2ARKCT peptide mediates inhibition of Gxcex2xcex3 function in vivo and that, in smooth muscle cells, that inhibition is associated with a modulation of cell proliferation.
It is a general object of the invention to provide a method of inhibiting smooth muscle proliferation.
It is a specific object of the invention to provide a method of inhibiting uncontrolled smooth muscle cell proliferation by inhibiting Gxcex2xcex3-signaling.
It is another object of the invention to provide a method of reducing intimal hyperplasia following vein grafting and restenosis following arterial injury.
The foregoing objects are met by the method of the present invention which comprises introducing into smooth muscle cells at a body site an agent that inhibits Gxcex2xcex3-mediated processes and thereby inhibits proliferation of the muscle cells. In one embodiment, the agent comprises a nucleic acid encoding a polypeptide corresponding to the Gxcex2xcex3-binding domain of xcex2ARK. In accordance with this embodiment, the nucleic acid is introduced into the cells in a manner such that the polypeptide is produced and proliferation of the smooth muscle cells is inhibited.
Further objects and advantages of the invention will be clear from the description that follows.